DOT1L is required for MLL-AF9–mediated leukemogenesis in vivo. (A) Diagram of the BMT procedure to examine the effect of DOT1L depletion on the establishment of AML by MLL-AF9–transformed cells in vivo. TAM treatment began at 3 weeks after transplantation. (B-G) In vivo deletion of DOT1L prevents MLL-AF9–mediated acute leukemia development. (B) Kaplan-Meier plot showing survival of transplanted mice treated and untreated with TAM. Median survival of wt/wt MLL-AF9–transplanted mice without TAM = 7 weeks (n = 17) and wt/wt MLL-AF9 transplanted mice treated with TAM = 7 weeks (n = 14). All 2lox/1lox MLL-AF9–transplanted mice treated with TAM (n = 18) survive at least 24 weeks after transplantation. (C) Representative image of spleens harvested from transplanted mice at 6 weeks after transplantation show splenomegaly in wt/wt MLL-AF9 transplanted mice (n = 3 per group). (D) Representative H&E staining of liver tissue sections at 6 weeks after transplantation show leukemic infiltration in wt/wt MLL-AF9 transplanted mice (n = 3 per group). (E) Donor cells depleted of DOT1L are absent in recipient bone marrow. Representative images showing FACS analysis of bone marrow cells isolated from transplanted mice (n = 3 per group). Donor MLL-AF9 cells are Ly5.2⁺ (blue) while recipient/protector cells are Ly5.1⁺/Ly5.2⁺ (red). (F) Percentage of MLL-AF9 LCs present in recipient bone marrow at 6 and 9 weeks after transplantation as determined by FACS analysis (n = 3 per group). (G) No transformed donor cells are detected by FACS analysis of peripheral blood obtained from 2lox/1lox MLL-AF9+TAM mice at 24 weeks after transplantation. Representative image from 1 of 6 mice analyzed.